Improved methods of isolation using affinity chromatographic procedures together with compositional studies of the following bacterial selenoenzymes are under investigation: glycine reductase selenoprotein A, Methanococcus vannielii formate dehydrogenase, acetoacetyl-CoA thiolase of Clostridium kluyveri and nicotinic acid hydroxylase of Clostridium barkeri. No selenocysteine residues have been detected in the polypeptide portions of the two latter selenoenzymes. Instead, derivatization procedures result in separation of Se from these proteins in a form that is as yet unidentified. Seleno tRNAs that contain bases specifically modified with selenium have been purified from C. sticklandii and M. vannielii and analyzed. Nuclease digests of tRNAs from both these organisms contain a Se-nucleotide that is less acidic than 4-thiouridylic acid and is thus differentiated from 4-selenouridylic acid.